CN1060178C - Derivatives of structurally modified vip and pharmaceutical compositions containing them - Google Patents

Abstract

The present invention provides a novel compound for treating male impotence. The compound belongs to a vasoactive intestinal peptide (VIP) and VIP <7-28> VIP<16-28> sheet, wherein a natural amino acid sequence is modified by using other natural or artificial amino acid to replace an amino acid residue at 5, 17 and 19 position. The compound has at least one tail end lipophilic group. A modified VIP sequence and a segment are prepared by a general peptide chain assembly method. The novel compound and a composition containing the novel compound are suitable for skin exposure application.

Description

The application of new structurally-modified active intestines peptide derivant, its pharmaceutical composition and treatment impotence thereof

The present invention relates to novel derivative, its pharmaceutical composition and the application aspect the treatment impotence of structurally-modified active intestines peptide (VIP).Especially relate to the synthetic and transdermal administration of the VIP conjugates of modification, contain the lipophilic end group, be used for raising property vigor and erection at conjugates described in the suitable composite ointment.

Bibliography of the present invention has: 1. Said, S. and Mutt, V., Nature 225,863 (1970) .2.Anderson, P.O.et al., J.Physiol.350,209 (1984) .3.Otteson, B.et al., British Medical J.288,9 (1984) .4.Dixon, A.F.et al., J.Endocro.100,249 (1984) .5.Gu, J.et al., Lancet 315 (1984) .6.Gozes, I.et al., Endocrinology 125,2945 (1989) .7.EP-0 354 992 A2; U.S.5,147,855,8.Wagner, G.and Gerstenberg, T., World, J.Urol.5 171 (1987) .9.US 4,605,641.10.Merrifield, R.B., J.Am.Chem.Soc.85,2149 (1963) .11.Steuart, J.M.and Young, J.D., Solid Phase Peptide Synthesis, Pierce

Chemical Corp．，Rockford，Ill．，1984 (2nd edition)．12． Okumura，M．et al．，Chen．Pharm．Bull．37，1375 (1989)．13．Sachs，B．D．in "Hormones and Behaviour in Higher Vertebrates"

(Eds．Balthazart，J．Prove，E．and Gilles，R．)Springer-Verlag Berlin

Heidelberg(1983)p．86．14．Wemer，H．et al．，Biochem．Biophys．Res．Commun．133，228 (1985)．

Impotence is a kind of influence syndrome widely of 10-15% male sex's population at least.According to estimates, only the U.S. just has 10,000,000 above men to suffer from various degree impotence disease.Theoretically, because the neuroendocrine relevant with aging decline, the man of any age above 40 years old may experience impotence once in a while.

Impotence can be produced by problem psychology or physiological, the suffering of illness that it can make patient or the people around them stand life.Another problem relevant with impotence is that the patient does not usually actively go to cure, particularly consider the doctor the obtainable limited medicine that makes things convenient for.The treatment pattern of the impotence that is caused by the organ reason except that the level and smooth muscle relaxant drug of injection such as Papaverine or phenoxybenzamine, also relates to surgery and transplanting.

With graft of penis treatment impotence a lot of shortcomings are arranged, this treatment needs surgical operation, and produces complete and irreversible erection tissue damage, and this damage hinders penis normal erection ability in the future.

Often cause priapism with Papaverine or phenoxybenzamine treatment impotence, (generally be several hours to twenty four hours) keeps erection over a long time.Except the issuable embarrassed look of the unusual projection of penis, it usually is painful and may irreversibly damages penile tissue.In order to alleviate, may need medicament adjusting in some cases.Even priapism does not take place when using Papaverine, this use also produces the misery as the burn in two minutes in the injection back, and demonstrated heavily clothes use Papaverine cause extensively in the vesicular fibrosis.Phenoxybenzamine is under a cloud in addition is carcinogens, thereby can not think that it is to the impotence active drug in the future.

As seen take a broad view of above-mentionedly, it is a kind of careful, effectively and the impotence methods of treatment of self control to need.From treatment be self control and effectively and not need the fact that gets involved in any surgical operation, transdermal uses medicine to seem to provide a kind of attractive method that is used for this treatment.Can not cause priapism with medicine of the present invention and medicine composite for curing.

Erection mechanism needs endocrine system, neural system, vascular system is intact.Vasoactive intestinal peptide (VIP) is at first isolated (1) by S.Said and V.Mutt, and demonstrates physiologically active widely, and it is found the neurohumoral several standards of erection (2) of regulating that reach in the recent period.VIP is detected in the fibrous porous unstriated muscle that innervates, and it raises during erecing (3,4), reduces in the impotence male sex (5).Inject external source VIP in addition and can cause male penis erection (3).The nearest demonstration, system's injection VIP energy pungency behavior (6) in the mouse that reduces male potential by test, VIP, VIP _7-28And VIP _15-28But segmental lipophilic derivative transdermal uses to improve sexual behaviour (7).In a experiment, show, do not make these male mouses be subjected to sexual stimulus, the VIP of 20 μ g nearly is expelled to only can causes the knuckle that penis is slight in the cavernous body (Corpus Cavernosum) of common male mouse and can not erect normal animal subject.But when combining, the few like this VIP of 1 μ g is expelled to just impels penis to erect fully in the cavernous body with sexual stimulus.

Also have known VIP analogue (9), some amino acid of wherein natural VIP sequence are by other replacement, and 1 Histidine wherein, and so-called N-end can arbitrarily be acetylation, and these compounds that do not appear in the newspapers can be strengthened the male sex's sexual behaviour.

The open 4-59794 of the Japanese Patent of publishing on February 26th, 1992 has described by drug administration by injection treatment impotence and has prevented a kind of by L-leucine-17-VIP-HSe-NH of segmental bronchus contractura ₂The VIP amidated homoserine derivative of the modification of expression.

The improved medicament and the composition that the purpose of this invention is to provide the skin-penetrating therapeutic impotence.

VIP, a kind of preface of 29 amino acid peptides is as follows:

1 7H-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-

16-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-

28 -Ser-Ile-Leu-Asn-NH ₂

The present invention is based on and is surprised to find, and compares with the natural VIP that has identical lipophilic end group, and the structurally-modified VIP that has at least one suitable lipophilic end group has tangible enhancement aspect the transdermal medication treatment impotence.Thereby the less relatively aminoacid sequence of stearyl VIP (St-VIP) changes and has caused bioactive increase.The VIP that further finds to have the modification of at least one lipophilic end group very at large constitutes the medicine that the skin-penetrating therapeutic impotence haves a great attraction.And, when using, use the VIP of the radiolabeled and modification that has identical lipophilic end group to estimate that its tissue infiltration increases with identical amount.

According to the present invention, the same discovery has the VIP of the modification of at least one lipophilic end group _7-28And VIP _15-28Fragment has obvious enhanced effect than the unmodified fragment that has the same end group accordingly, and this is because the improved penetrating power of organizing.

Find that further some carrier can increase organizes osmosis, makes said VIP structurally-modified, that have the lipophilic end group and VIP structurally-modified, that have the lipophilic end group _7-28Or VIP _15-28Segmental pharmaceutical composition becomes by the transdermal administration mode treats the impotence more efficient drug.

The invention provides new material, this material be have formula I sequence modification vasoactive intestinal peptide (VIP)/or the modification VIP of formula I sequence _7-28Or VIP _15-28Segmental conjugates, and the functional deriv of any this conjugates,

1 7R ¹-Y ¹-His-Ser-Asp-Ala-X ¹-Phe-Thr-Asp-Asn-Tyr-

16Thr-Arg-Leu-Arg-Lys-Gln-X ²-Ala-X ³-Lys

28Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH-Y ²-R ²In the formula I

R ¹And R ²Identical or inequality, they each hydrogen, saturated or undersaturated lipophilic group or C naturally ₁-C ₄Alkyl or carboxylic acyl group, and R ¹And R ²In at least one be lipophilic group;

Y ¹And Y ²Identical or different, they each naturally-CH ₂-or-CO-, or a key is (as relative R ¹Or R ²When being hydrogen);

X ¹, X ²And X ³Identical or different, if their each residues natural naturally or alpha-non-natural amino acid are X ¹And X ³When all being Xie Ansuan, X ²Can not methionine(Met).

Any said functional deriv is the compound that has functional group at least one amino-acid residue of 2-27 position, and they are the conjugatess that produce biologic activity.

X ¹, X ²And X ³Preferably by the lipophilic amino-acid residue of leucine, Isoleucine, nor-leucine, Xie Ansuan, tryptophane, phenylalanine and methionine(Met) representative.

The particularly preferred compound of the present invention is X among the following formula I ²Be nor-leucine residue, X ¹And X ³Each is Xie Ansuan, Y naturally ¹Be-CO-and R ₁Be C ₁₇Those compounds of alkyl.

The most handy solid-phase synthesis of the peptide chain of new compound of the present invention preparation (10,11), in case these peptide chains be assembled, even connect non-hydrogen end group R ¹Y ¹-and/or R ²Y ²-.

Work as Y ¹And/or Y ²When being carbonyl, promptly compound has terminal R ¹CO-and/or R ²During CO-, R ¹CO-and R ²CO-both one of or both all can be introduced into by acidylate step commonly used.

At preparation Y of the present invention ¹Be-CH ₂-compound, promptly have terminal R ¹-CH ₂-compound the time, R ¹-CH ₂-Ji can with known method already at first by with aldehyde R ¹The CH=O coupling restores resulting R ¹-CH=N-group is introduced into.Has terminal R ²-CH ₂-compound can be by using formula R ²-CH ₂-NH ₂Amine fracture peptide obtain.

The present invention also provides the pharmaceutical composition of transdermal administration with treatment boar impotence, and said composition comprises that above-mentioned formula I compound is as active ingredient and suitable pharmaceutically acceptable carrier.

Carrier is preferable over those carriers that can improve the infiltration of active ingredient tissue.The example of suitable carrier has glycerine, lubricant, pannonit and Sefsol ^TMAnd their mixture.Sefsol is trade mark (the Nikko Chemicals of Monooctamoin, propylene glycol dicaprate, propylene glycol dicaprylate, tricaprylin and anhydro sorbitol list octanoate, Tokyo), they are preferred vectors of the present composition, and wherein, Monooctamoin is particularly preferred.

The present invention also provides the transdermal dispenser of the particular types of lasting release yoke compound, and it comprises a medicator that is suitable for dermal administration, and said conjugates wherein is housed.

If desired, the conjugates in the medicator can be made into the special medicaments composition.

New compound of the present invention is useful in the treatment of impotence, particularly by the transdermal administration mode.This methods of treatment demonstrates the several advantages above prior art.One, it is non-surgery, and does not stay tissue injury, moreover it does not cause priapism relevant with other medicines or Burning Pain sense.To compare also be a kind of careful and mode of administration easily in (intracavernosal) injection in transdermal administration and the cavernous body.And this method can be used a kind of continuously slow releasing device, and this device can make spontaneous sexuality carry out and not need long-time preparation, has removed the many common embarrassed looks of patient like this from.

Will be with reference to the accompanying drawings in following detailed description, wherein:

Figure 1A, 1B, 1C and 1D show the biological test result of bulbocavernous reflex described in the embodiment 6;

Fig. 2 and Fig. 3 show dermal osmosis result of experiment described in the embodiment 7.

The following example illustrates many aspects of the present invention, should be appreciated that to the invention is not restricted to this.Embodiment 1: stearyl-Nle ¹⁷-VIP's is synthetic

Said new the synthetic of modification VIP conjugates will be by most preferred of the present invention, i.e. stearyl-nor-leucine ¹⁷-VIP (St-Nle ¹⁷-synthesizing and illustrate VIP).

Peptide chain according to the General Principle of Merrifield solid phase method (10,11) on methyldiphenyl methylamine resin (MBHA) (buying) from Switzerland Nova company, manual setting in mechanical vibrator.All solvents, methylene dichloride (CH ₂Cl ₂), N-Methyl pyrrolidone (NMP) and methyl-sulphoxide (DMSO) all be AG products of German Merck company.The N of trifluoroacetic acid (TFA), diisopropyl ethyl amine (DIEA), N '-dicyclohexylcarbodiimide (DCC) buys from U.S. Aldrich company.I-hydroxybenzotriazole (HOBT) obtains from Switzerland Nova company.All protected amino acid derivative (Boc-AA) all are L-configurations, all are to obtain from Switzerland Bachem company.In whole synthesizing, N a-amino acid functional group protects with tertiary butyl oxygen carbonyl (t-Boc).The following protection of side chain functionalities: Ser, Asp, Thr benzyl protection; Lys 2-benzyl chloride base oxygen carbonyl-protection; Tyr is with 2, and the 6-dichloro benzyl is protected; His benzyl oxygen carbonyl-protection; Arg protects with p-toluenesulfonyl.

From using DCC (0.42g; 2mmol) and HOBT (0.272g 2mmol) makes reagent with Boc-Asn (0.46g; 2mmol) be coupled on the methyldiphenyl methylamine resin (1g) and begin to synthesize.Loading level (0.39mmol/g) is measured by amino acid analysis.Unreacted residual is amino on the polymkeric substance passes through at CH ₂Cl ₂React capping with aceticanhydride and triethylamine (being respectively 1ml and 0.5ml) (10ml).The peptide chain assembling begins with the Boc-Asn-MBHA resin by the scheme shown in the table 1.

Table 1-solid-phase peptide is synthetic

Step	Agent/solvent	Time (minute)
			1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16	TFA is at CH 2Cl 2In (30%V/V) TFA at CH 2Cl 2In (50%V/V) CH 2Cl 25%DIEA is at CH 2Cl 2(V/V) 5%DIEA (V/V) NMP ninhydrin test 1.6mmol Boc A.A+ 1.6ml1N HOBT+1.6ml 1NDCC in NMP all activates-30 minutes in advance in NMP; Filtration also adds solution to polymkeric substance, DMSO (1g), (final volume 20%V/V) DIEA-, (6mmol is in NMP) NMP CH2Cl 2Ninhydrin reaction 10%Ac 2O+5%DIEA is at CH 2Cl 2Middle 10%Ac 2O is at CH 2Cl 2Middle CH 2Cl 2	3 17 5×2 5 2 5×2 45 20 10 5 3×2 5 3×2

The solvent volume of all washings and reaction is all measured the 10ml/g resin.All couplings all use the HOBT active ester of Boc-amino acid derivative to finish, and this active ester is prepared by DCC before each coupling step.Mol ratio is that 4: 1 Boc-amino acid/11-hydroxybenzotriazole ester (Boc-AA-OBT) and the alpha-amino group that increases peptide chain are respectively applied for coupling.Coupled reaction is by boiling monitoring in 2 minutes with several mg (about 3mg) polymkeric substance in the pyridine-aqueous solution of triketohydrindene hydrate.The amino acid whose coupling of Boc repeats secondary and reacts completely with assurance.In the coupling second time, use the Boc-AA-OBT of half amount, after generally each coupling step was finished, residual amino was by the dichloromethane solution process resin capping with aceticanhydride (10%) and diisopropylethylamine (5%).

After the peptide chain assembling is finished, removing the t-Boc protecting group of His, generally is to be used in CH ₂Cl ₂In 50%TFA, the alpha-amino group that of newly dissociating is with DCC (0.42g; 2mmol) and HOBT (0.27g; 2mmol) make reagent and stearic acid (0.37g; 2mmol) coupling.Reaction was carried out 45 minutes and was repeated secondary.Peptide-resin of finishing assembling is by scheme CH ₂Cl ₂Washing is then at P ₂O ₅Last vacuum-drying is spent the night.Make the protecting group deblocking and peptide is ruptured from resin with the anhydrous HF technology.(1: 1, V/V) existence was down at the Teflon that 9ml HF is housed in that 1.5ml toluene-and p-cresol mixture are arranged for peptide-resin (1g) ^TMHandled 1 hour down at 0 ℃ in the HF device (polypeptide system).Vacuum is removed HF, resin with the ether of no superoxide handle, filter, with the ether washing, dry and be used in 50% acetate in the water (3 * 25ml) extract.To contain the filter liquor freeze-drying and obtain stearyl-Nle ¹⁷-VIP crude product (400-500mg).

Dissolving crude product in 50% aqueous acetic acid, cross Sephadex G-25 post (75 * 2cm), make eluent with 0.1N acetate.Be eluted under the 274nm and monitor with spectrophotometry.Collect the peak of first appearance, with aqueous solution freeze-drying, obtain the not having fragrant additive peptide of (in HF-fracture step, adding) as scavenging agent.Productive rate is 50-70%.

With high performance liquid chromatography (HPLC) method purifying Sephadex-fraction products.Can certainly carry out this purifying to thick peptide.Purifying can be used Merck RP-8 post (7 μ M; 250 * 25mm) finish.The peptide of use in 10% acetonitrile solution, linear gradient elution peptide between 75% acetonitrile solution of the 0.1%TFA aqueous solution and 0.1%TFA, flow velocity 5ml/min.Collect each cut, after analyzing the HPLC monitoring, discard useless component (cuts made), merge gained cut and freeze-drying.The productive rate of pure peptide is 30-35%.

After carrying out acid hydrolysis completely (6NHCl), by analyzing HPLC (Merck RP-8,125 * 10mm post) and amino acid analysis, determine the purity of product, it can provide respectively forms amino acid whose expected value.

Hard ester acyl-Nle ¹⁷-VIP is more stable than stearyl-VIP, and aminoacid sequence does not wherein change.Other lipophilic derivative is coupled to Nle with identical step ¹⁷On-the VIP, wherein after removing the t-Boc protecting group of His, new free amino is coupled on the suitable carboxylic acid with DCC and HOBT as stated above.Embodiment 2: stearyl-Nle ¹⁷-VIP's is synthetic

St-Nle ¹⁷Following the finishing of another kind of synthetic method of-VIP:

Peptide chain according to Merrifield solid phase general theory of law 4-(2 ', 4 '-dimethoxy phenyl-aminomethyl)-phenoxy resin (buying) from Switzerland Nova company on, manual setting in mechanical vibrator.All solution: methylene dichloride (CH ₂Cl ₂), N-Methyl pyrrolidone (NMP) and dimethyl formamide (DMF) all be AG products of German Merck company.Trifluoroacetic acid (TFA), diisopropyl ethyl amine (DIEA) and N, N '-dicyclohexylcarbodiimide (DCC) buys from U.S. Aldrich company.I-hydroxybenzotriazole (HOBT) obtains from Switzerland Nova company.All protected amino acid derivative (FMOC-AA) all are L-configurations, all obtain from Switzerland Bachem company.In individual synthesizing, N ^α-amino acid functional group protects with fluorenylmethyloxycarbonyl (FMOC).The following protection of side chain functionalities: Ser, Asp, Thr protect with the tertiary butyl; Lys protects with tertbutyloxycarbonyl; His protects with benzyloxymethyl (BOM); Arg protects with methoxyl group Three methyl Benzene alkylsulfonyl (Mtr).

Synthetic 1 and 2 (referring to schemes) set by step are by from the commercial polymer, promptly 4-(2 ', 4 '-dimethoxy phenyl-FMOC-aminoethyl) (remove the FMOC group on the 0.47mmol amino/g) begins phenoxy resin.Use is contained in 2 10g polymkeric substance in the reaction vessel.The solvent volume of using in each container is 20-25ml.The assembling of peptide chain from DCC (0.84g, 4mmol) and HOBT (0.55g, (0.92g 4mmol) is coupled on the resin (5g) and begins with FMOC-Asn 4mmol) to make reagent.Repeat the coupling step.Loading level (0.39mmol/g) is measured by amino acid analysis.Unreacted residual on the polymkeric substance is amino to be passed through at CH ₂Cl ₂In with aceticanhydride (10%) and diisopropylethylamine (5%) reaction capping.Peptide chain assembles by the scheme shown in the table 2 from the FMOC-Asn-resin.

Table 2--solid-phase peptide is synthetic

Except that coupling (the volume of the 5ml/g resin that uses during step 10), the volume of all washings and reaction soln is all measured the resin to 10ml/g, all couplings all use the HOBT active ester of FMOC-amino acid derivative to finish, and this active ester is prepared by DCC before each coupling step.Mol ratio is that 2: 1 FMOC-amino acid/11-hydroxybenzotriazole ester (FMOC-AA-OBT) and the α amino that increases peptide chain are respectively applied for coupling.Coupled reaction is by boiling monitoring in 2 minutes with several milligrams of (about 3mg) polymkeric substance in the pyridine-aqueous solution of triketohydrindene hydrate.The amino acid whose coupling of FMOC-repeats twice or more times is to guarantee complete reaction.In the coupling second time and during other necessary couplings, use the FMOC-AA-OBT of half amount.Be intended to add that next amino acid whose subsequent step only could begin after negative ninhydrin reaction (step 15 is referring to scheme).After general each coupling step was finished, residual amino was by coming capping with the dichloromethane solution process resin of aceticanhydride (10%) and diisopropylethylamine (5%).

After the peptide chain assembling is finished; remove the FMOC protecting group of His, generally be by the piperidines in DMF and newly dissociate the alpha-amino group that with DCC (0.84g, 4mmol) and HOBT (0.54g; (0.74g 4mmol) gets on to stearic acid 4mmol) to do reagent coupling (in each reaction vessel).Reaction continued 120 minutes and repeated twice.The resin that contains the peptide chain that assembles fully is by scheme CH ₂Cl ₂Washing is then at P ₂O ₅Last vacuum-drying is spent the night.Make the protecting group deblocking by the following method and peptide is ruptured from resin; The 1g dried resin is placed the 100cc flask, and to wherein adding thioanisole (2ml) and dithioglycol (2ml).This mixture is cooled to 4 ℃ in ice bath, add the 20ml trifluoroacetic acid, adds trifluoromethanesulfonic acid (2ml) after 5 minutes again, and this mixture was stirred 50 minutes gently in room temperature.

This reaction mixture is cooled to 4 ℃, pours in the 500ml dry diethyl ether., on sinter funnel, solid matter (resin and peptide) is filtered after 60 minutes 4 ℃ of stirrings,, use 50% acetic acid aqueous solution (100ml) extraction then with dry diethyl ether washing, drying.The solution that gained contains peptide concentrates under high vacuum, and residue (about 15ml) is directly gone up SephadexG25 post (45 * 6cm).With this post of 0.1N acetate wash-out, flow velocity is 45ml/ hour.Elutriant is monitored under 274nm.The water-soluble liquid cooling that will contain required cut is done, obtain the not having fragrant additive peptide of (adding as scavenging agent in acid (acidolytic) cleavage step).Product is the white powder of about 400mg.

Amino acid analysis after the thorough hydrolysis of acid has shown aminoacids content and the ratio that this material is required.

(HPLC) is further purified the Sephadex-fraction products by high performance liquid chromatography (HPLC), can certainly carry out this purifying to the crude product peptide.Purifying can use Merck RP-8 post (7 μ m, 250 * 25mm post) to finish.The peptide of use in 35% acetonitrile solution, linear gradient elution peptide between 75% acetonitrile solution of 35% acetonitrile and the 0.1%TFA aqueous solution and 0.1%TFA, flow velocity 10ml/ branch.Collect each cut, after analyzing the HPLC monitoring, discard useless component.Merge gained cut and cold doing.The productive rate of pure peptide is 30-35%.Embodiment 3:R ¹-CH ₂-Nle ¹⁷The preparation of-VIP derivative

As described in embodiment 1, peptide chain is at polymer support, and promptly formyl radical (methoyl) benzhydrylamine resin (MBHA) (containing 0.39mmol Asn/1gr) is gone up assembling.After last amino-acid residue (Histidine) connects, remove N-α-protecting group (t-Boc) with TFA, polymkeric substance is handled with DIEA, washs and carries out ninhydrin reaction.Polymer suspension adds corresponding aldehyde R in ethanol (1gr/10ml) ¹-CH=O (3-4 equivalent aldehyde is to 1 equivalent free terminal amino group), mixture stirs gently in room temperature and spends the night.Filter polymkeric substance, and the usefulness washing with alcohol (3 * 10ml), be suspended in ethanol and NaBH again ₄In (3-4 equivalent reductive agent is to 1 equivalent Schiff alkali; R ¹-CH=N-His ...-), mixture at room temperature stirred two hours gently.Perhaps (in the presence of 0.1-0.2ml acetate) uses NaBH ₃CN (the 3-4 equivalent is to 1 equivalent Schiff alkali).Condensation and reduction reaction also can be carried out in other organic solvent.These solvents are for example DMF or NMP.After reduction is finished, filter polymkeric substance, washing, drying, and and stearyl-Nle ¹⁷-VIP equally handles with HF.With with method purifying crude product identical described in the embodiment 1, obtain required final product.

After carrying out acid hydrolysis completely (6NHCl), determine the purity of product by analyzing HPLC (Merck RP-8,125 * 4mm post) and amino acid analysis, it can provide the amino acid whose expected value of each component.

The stearyl analogue of following VIP also can use method for preparing: St-Leu ⁵, Nle ¹⁷-VIPSt-Leu ¹⁷-VIPSt-Leu ⁵, Leu ¹⁷-VIPSt-Thr ⁵-VIP

Can use and above-mentioned stearyl-Nle ¹⁷Analysis HPLC and amino acid analysis method that-VIP is the same are determined product purity, provide the amino acid whose expected value of each component.Embodiment 4:R ¹-Y ¹-NH-Nle ¹⁷-VIP-NH-R ²Preparation

First amino acid Boc-Asn be connected to as follows 1% crosslinked chloromethylated polystyrene (Chemalog, South Plainfield, N., J., USA) on: to the amino acid derivative (5mmol in raw spirit (35ml); 1.16gr) the middle triethylamine (4.75mmol that adds; 0.66ml), mixture was placed 5 minutes in room temperature.Add polymkeric substance (5gr), mixture slowly refluxed 60 hours at 78 ℃.Another kind method is, 5mmol Boc-Asn is dissolved in the mixture of EtOH (12ml) and water (3ml), uses 20%C _sCO ₃The aqueous solution is transferred pH to 7.5.Solution is used P with benzene flash distillation three times in moisture eliminator ₂O ₅Dry residue 5 hours.Add DMF (30ml) with lysate, then add the 5gr polymkeric substance, mixture stirred 36 hours at 50 ℃.Loading level (0.4mmol/gr) is measured by amino acid analysis.

Assemble with the embodiment 1 the same peptide chain of finishing, only be to use Boc-Asp (β-cyclohexyl ester) to replace Boc-Asp (β-benzyl esters).Cyclohexyl is separated more stable to ammonia.In case finish required peptide chain assembling, with above-mentioned the same washing and dry polymer.Product be suspended in the dehydrated alcohol or 1: 1 V/V mixture of EtOH and DMF (1gr/10ml) in, then, add corresponding amine (R ²-NH ₂20mmol), stirred this mixture under the room temperature gently 48 hours.Use solvent systems N-butanols: acetate: H ₂O: (15: 3: 12: TLC 10V/V) showed and isolating product from the polymer support occurred pyridine.Polymkeric substance with ethanol (3 * 10ml), DMF (3 * 10ml) extraction, solvent evaporates under high vacuum.Then, with aforementioned the same, handle the semi-solid residue of oily to remove Side chain protective group with HF.Crude product is used and stearyl-Nle ¹⁷Described identical method of-VIP and suitable productive rate purifying obtain required end product.

Embodiment 5: composite ointment

Prepare following composite ointment, and said composition is carried out the test of the modification VIP derivative that transdermal administration lipophilic yoke of the present invention closes.The similar conjugates of unmodified VIP derivative also tested be used for comparison.

1. contain the paste of glycerine as carrier:

Prepare this paste with the following method: 2g glycerine+10mg stearyl-VIP+7 drips DMSO (dimethyl sulfoxide (DMSO)).Every mouse is received in the 30-50 μ g stearyl-VIP (GLY among Fig. 1) in about 10 μ l paste.

2. contain the paste of lubricant as carrier:

Prepare paste with as above method, just replace glycerine with lubricant (K-Y LubricatingJelly, Johnson﹠Johnson contain propylene glycol and glycerine), 7 DMSO equal about 130 μ l (LUB among Fig. 1).

3. contain lubricant and pannonit paste as carrier:

The same with above-mentioned (2), with 1.7ml pannonit (1mg/ml) preparation, (LUB-NTG among Fig. 1).

4. contain Sefsol 318 ^TM(12) as the paste of carrier:

This composite ointment is: 31.2 μ l 10%Sefsol 318 ^TM(Monooctamoin), the 0.24mg stearyl-VIP in 31.2 μ l DMSO (every animal 1-2 μ l) (SEFS among Fig. 1) or the 0.24mg stearic acid-Nle in 31.2 μ lDMSO ¹⁷-VIP (SEFS* among Fig. 1).Embodiment 6: the biological test of bulbocavernous reflex effect

Biological test carries out bulbocavernous reflex mensuration to the castrating mouse after relating to the VIP conjugates transdermal administration modification or natural.In first kind Bioexperiment, measured of the effect of the composition of stearyl VIP and various carriers to bulbocavernous reflex, find that Sefsol is the most effective carrier.Stearyl-VIP and Sefsol 318 have been compared in another kind of experiment ^TMBonded effect and stearyl-Nle ¹⁷-VIP and Sefsol 318 ^TMThe bonded effect.

In the second class Bioexperiment, monitored the distribution of several radiolabeled VIP derivative in various organs.(a) method

The impotence animal model

Use is because of the mouse that castrating has reduced property potential, and male mouse (250-300g, about 3 monthly ages) was in 12 hours bright, 12 hours dark cycles, and experiment is always in dark period, and carried out in 2-6 hour the dark back of arriving.Male mouse is castrated, with the form replacing section testosterone (4 μ g/100g body weight) of injection continuous 14-21 days (experimental session) every days.A week experimentizes after the surgical operation.

The direct appraisal of bulbocavernous reflex (erection)

Adopt a kind of program of measuring penis sexual reflex technology of using, it can directly appraisal erection after the transdermal medication.Most of successful regeneration depends on the accurate execution of behavior unit temporary transient formation, that function is relevant.In these experiments, we concentrate the terminal stage (redden and the consequent of penis are finished the expansion and elongation of erection) of research erection process and monitoring to the first time E2 with the latent period that becomes cup-shaped (CUP) (13) first time.

In order to test, to be used in the method for its health previous section of sealing in the loose suitable cylinder (7cm diameter) and will every animal to be limited in but clinostatism is put.Behind the tight trunk of belt bolt, glans penis is extruded from its sheath, and the thin wooden medicator that is installed in the penis rear portion is clamped gently perpendicular to belly.The leg observed person of male mouse clamps, and this position keeps at duration of test always.Whole process is 45 minutes.Record is with plotting E2 and become hiding and number of cup-shaped.

E2 is defined as the pure erection fully that then forms cup (wherein the penis end becomes the structure of cup sample, and gland opens, make penis terminal portions thicker than its foundation part).This terminal stage needs E2, and it may be necessary (13) before the ejaculation.Use all parameters, people can obtain the reliable measurements of tested mouse property vigor.(b) result

Topical application contains stearyl-VIP (St-VIP) and stearyl-Nle ¹⁷-VIP (St-Nle ¹⁷-VIP) different composite ointments are shown in Figure 1A and 1B to the effect of bulbocavernous reflex.

In this example relevant, 6 to 10 animals are tested with each variable.Control animals received does not have St-VIP or St-Nle ¹⁷The composite ointment of-VIP.Figure 1A: to hiding of the E2 first time

Shown in Figure 1A, when use contains at Sefsol 318 ^TMIn St-Nle ¹⁷Observe the shortest the hiding to the E2 first time during composition of-VIP (SEFS* in the 5th road), the E2 of this composition is hidden is about 1 minute.Figure 1B: to becoming for the first time hiding of cup-shaped

Figure 1A for another example, at transdermal administration at Sefsol 318 ^TMIn St-Nle ¹⁷Observe behind-the VIP the shortest the hiding that become for the first time cup-shaped (SEFS* in the 5th road, the shortest hiding=10 minute), the number of Fig. 1 C:E2

Consistent with Figure 1A and 1B, when being used in Sefsol 318 ^TMIn St-Nle ¹⁷Observe the highest E2 number (SEFS* in the 5th road) during the combination treatment mouse of-VIP.Use the E2 number of this composition to use stearyl-VIP and Sefsol 318 ^TMComposition (SEFS* in the 4th road) time viewed E2 number much higher.These results obviously show the advantage (34 couple 24 of duration of test E2) of structurally-modified VIP conjugates.Fig. 1 D: the number that becomes cup-shaped

With above-mentioned respectively scheme consistent, to containing at Sefsol 318 ^TMIn St-Nle ¹⁷The composition of-VIP is also observed into the highest number (SEFS* in the 5th road) of cup-shaped.Sefsol 318 ^TMWith St-VIP and St-Nle ¹⁷The one-tenth cup-shaped number that-VIP is used in combination is respectively 12 and 20 (relatively the 4th and the 5th roads).

The Main Conclusions of all these experiments is: the variation that the aminoacid sequence of stearyl-VIP is relatively little (promptly replacing Met at 17 with Nle) has increased the effectiveness of this medicine irritation vigor greatly.

Second conclusion is: Sefsol 318 ^TMBe transdermal release stearyl-VIP and stearyl-Nle ¹⁷The only carrier of-VIP.By all four test parameters, transdermal administration contains Sefsol 318 ^TMWith stearyl-Nle ¹⁷The composition of-VIP is than Sefsol 318 ^TMMore effective with the composition of stearyl-VIP.Embodiment 7: distribute in dermal osmosis experiment and the body

These experiments are divided into two groups.In first group of experiment, we have finished the radiolabeled stearyl-Nle of transdermal administration ¹⁷-VIP is (at Sefsol 318 ^TMIn) each organ time course of distributing in vivo.In second group of experiment, we have compared behind the transdermal administration and Sefsol 318 ^TMThe infiltration of 4 kinds of radio-labeled VIP derivatives together.To each data point, use 2-4 animal.Derivative with ¹²⁵I chlorine ammonia T method (the chloramine-T based method) mark (14) of alkalization.

In first group of experiment, stearyl-Nle ¹⁷-VIP uses ¹²⁵I is carried out radio-labeled.Every animals received 2.2 hundred ten thousand CPM in 6 μ l radio active materials+2 6 μ l paste (No. 4 paste among the embodiment 5).In two are independently tested, each time point is used two animals.For avoiding buccal absorption, the mouth of animal is sealed.Fixed time after topical application, animal is slaughtered, remove two parts of tissue samples, to weigh and count with gamma counter, measured tissue is: lung (LU), the heart (HE), kidney (KI), liver (LI) and intestines (IN).

The result who is shown in Fig. 2 clearlys show: peak value was released in after the administration between 15-60 minute, St-Nle ¹⁷-VIP disappeared after 2.5 hours.

In second group of experiment, prepared Sefsol 318 ^TMWith VIP, St-VIP, Nle ¹⁷-VIP and St-Nle ¹⁷The composition of-VIP.The experiment with Fig. 2 in the same finishing, only difference is every animals received 2.4 hundred ten thousand CPM.Each variable is used 2-4 animal.Animal was slaughtered in 30 minutes after the medication.

The result who is shown in table 3 is consistent with the result of sexual reflex example, as finding St-Nle ¹⁷The maximum concentration of-VIP composition, said composition are the most effective medicines.

The same with in the bulbocavernous reflex experiment, these last experiments also demonstrate at dermal osmosis and organize St-Nle aspect the distribution ¹⁷-VIP exceeds the advantage on other derivative.

Embodiment 8: toxicologic study

In order to estimate stearyl-Nle ¹⁷The toxic degree of-VIP and possible side effect are carried out two class researchs: 1. emergency toxicology research; 2. repeat administration toxicity research.

Medicine is prepared as follows basically: 539mg stearyl-Nle ¹⁷-VIP+2.31ml10%Sefsol+2.31ml40% Virahol.Laboratory animal is mouse (a 160-300g body weight).

In emergency toxicology research, three kinds of route of administration have been selected: 1. subcutaneous; 2. intravenous; 3. oral.Adopt subcutaneous and oral administration, when using the dosage (it be biological activity dosage 1000 times) of 7mg/ mouse, all do not find mortality, also do not observe pronounced side effects.Adopt intravenous injection, find LD ₅₀Be the 7mg/ mouse.In the repeat administration toxicity research, adopt local application's every day (to sexual organ), even when the dosage of 3.5mg/ animal, also do not observe mortality and any side effect.Generally speaking, these studies show that this material is nontoxic, and can be used for clinical trial.

Claims (14)

1. modification VIP in the conjugates of the vasoactive intestinal peptide (VIP) of the modification with formula I sequence or the formula I sequence _7-28Or VIP _16-28Segmental conjugates:

1 7R ¹-Y ¹-His-Ser-Asp-Ala-X ¹-Phe-Thr-Asp-Asn-Tyr-

16Thr-Arg-Leu-Arg-Lys-Gln-X ²-Ala-X ³-Lys

28Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH-Y ²-R ²In the formula I

R ¹And R ²Identical or inequality, they each hydrogen, saturated or undersaturated lipophilic group or a kind of C naturally ₁-C ₄Alkyl or carboxylic acyl group, condition is R ¹And R ²In at least one be lipophilic group;

Y ¹And Y ²Identical or different, they each naturally-CH ₂-or-CO-, perhaps as relative R ¹Or R ²It when being hydrogen a key;

X ¹, X ²And X ³Identical or different, if they each residue of natural amino acid naturally is X ¹And X ³When all being Xie Ansuan, X ²It or not methionine(Met).

2. conjugates as claimed in claim 1, wherein X ²It is nor-leucine.

3. as the conjugates of claim 1 or 2, X wherein ¹And X ³Each is Xie Ansuan naturally.

4. conjugates as claimed in claim 1, wherein R ¹Be C ₁₇Alkyl.

5. conjugates as claimed in claim 1, wherein Y ¹And Y ²In any one be-CO-R ¹-Y ¹And R ²-Y ²In any one be hydrogen.

6. conjugates as claimed in claim 5, wherein Y ¹And Y ²All be-CO-.

7. conjugates as claimed in claim 1, wherein Y ¹And Y ²In any one be-CH ₂-, R ¹-Y ¹And R ²-Y ²In any one be hydrogen.

8. conjugates as claimed in claim 7, wherein Y ¹And Y ²All be-CH ₂-.

9. stearyl-nor-leucine ¹⁷-VIP.

10. conjugates as claimed in claim 1 is the VIP of modification _7-28Or VIP _16-28Segmental conjugates.

11. the pharmaceutical composition of transdermal administration, said composition contain any one conjugates among the claim 1-9 as active constituent, this component can at random combine with pharmaceutically acceptable carrier.

12. as the pharmaceutical composition of claim 10, wherein said pharmaceutically acceptable carrier is selected from: glycerine, lubricant, pannonit, Monooctamoin, propylene glycol dicaprate, propylene glycol dicaprylate, tricaprylin and anhydro sorbitol list octanoate and their mixture.

13. as the pharmaceutical composition of claim 11 or 12, wherein said active ingredient is stearyl-nor-leucine ¹⁷-VIP, said pharmaceutically acceptable carrier is a Monooctamoin.

14. pharmaceutical composition with each conjugates preparation treatment impotence among the claim 1-9.

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